The overall goal of this procedure is to offer an alternative and inexpensive method to study inflammatory bowel disease or IBD using DY nitro benzene zonic acid or DNBS. This is accomplished by first intra rectally administering DNBS via gentle insertion to the anus. In the second step, tissue sections of the large intestine are acquired for histological screening and to assess the myelo peroxidase activity of the DNBS challenged tissues, ultimately the effects of dietary oils on weight loss and colitis can be evaluated by both macroscopic and microscopic methods.
The main advantage of this matter over the existing dextron sodium sulfate method is that DNBS method is easy to perform, less expensive, and highly reproducible. This method can help answer key questions in the IBD research field, such as what are the roles of stress and depression in the recurrence of IBD, or what are the underlying mechanisms in intestinal injury and repair For rectal installation of DNBS in mice begin by attaching a one milliliter syringe to a 19 gauge needle. Equipped with a polyethylene 50 catheter then aspirate freshly prepared DNBS into the catheter.
Next, apply tear gel to an anesthetized mouse and apply gentle pressure with the fingertips to the posterior end of the animal to remove any stool present in the distal colon. Now inject a small amount of the DNBS solution to lubricate the colon for easier insertion while gently inserting the catheter intra rectally about three to four centimeters. Once the catheter is in place, inject 100 microliters of DNBS or an equal volume of 50%ethanol for the control animals.
Then position the animal head down for 90 seconds to avoid loss of the reagents. After DNBS administration, feed the animals 8%sucrose water in 0.2%saline to prevent dehydration during the first week. Carefully monitoring the animal's daily for weight loss, dehydration, or distress for the duration of the experiment meant at the desired time point.
After DNBS challenge, open the abdominal cavity of the euthanized animal and remove the entire large intestine. Then open the intestine longitudinally. Macroscopic mucosal ulcers should be observed approximately two to three centimeters proximal from the distal end of the colon.
Collect 0.5 centimeter sections proximal to the site of maximal macroscopic damage in the experimental animals and segments of the colon from the corresponding anatomical locations from the control animals. Then snap freeze 0.5 centimeters from one control and 1D NBS treated section in liquid nitrogen for assessment of myelo peroxidase activity and fix the other sections in 10%neutral buffered formin overnight or histological analysis. The next morning, wash the formalin fixed tissues with 70%ethanol and place in a cassette to send to a histology facility to be embedded in paraffin and stained with hematin and eoin for histological scoring.
Then score at least three tissue sections from each animal under a light microscope to determine the histological damage scores for each group To assay the myelo peroxidase activity of the DNBS challenged tissues. First homogenize the snap frozen control and experimental tissues in myelo peroxidase homogenization buffer to give a 50 milligram colon segment per milliliter of homogenization buffer suspension. Then aliquot the homogenized solution into one milliliter portions and centrifuge them for two minutes at 10, 000 times g and four degrees Celsius.
Next, transfer 100 microliters of each aliquot to individual cuvettes and add 2.9 milliliters of PBS supplemented with IDE, dihydrochloride and hydrogen peroxide mixing vigorously. Finally, measure the rate of absorbance using a spectrophotometer at 460 nanometers. Following intra rectal challenge with DNBS as just demonstrated.
Significant weight loss is observed by 48 hours in rats supplemented with fish or canola oil, and by 72 hours in rats supplemented with safflower oil. The maximum weight loss observed occurs at 48 hours in fish oil, supplemented rats, and by 72 hours in rats. Supplemented with canola or safflower oil.
Weight gain resumed after 72 hours and by five days post DNBS administration, fish oil and safflower oil treated groups exhibited body weights similar to their baseline values. Control rats supplemented with the dietary oils and challenged with intra rectal ethanol steadily gained weight over the entire five day study period. The intestinal damage caused by DNBS can be widespread.
However, the area of maximal damage is generally localized to the distal six centimeters of the colon in rats and the distal three centimeters in mice. Upon closer histological assessment, extensive epithelial injury, including the formation of ulcers, neutrophil and lymphocyte infiltration, mucus depletion, and edema is observed. Although these histological signs of inflammation and tissue damage are evident in all three dietary oil supplemented groups upon challenge with DNBS, the most severe damage is seen in the safflower oil group followed by the canola oil group.
Whereas rats in the fish oil group demonstrate minimal epithelial injury and a limited infiltration of inflammatory cells into the mucosa cosa. Differences in the histological damage between the samples can be quantified to allow for a better comparison between the groups. The graph illustrates the histological damage scores for the three dietary oil groups during DNBS colitis.
As is observed in the h and e stained sections, the safflower oil supplemented group exhibits the greatest extent of damage followed by the canola oil group. While the fish oil group shows the least amount of damage, the extent of inflammatory cell infiltrate particularly of neutrophils can be further examined by performing a myelo peroxidase activity assay. As expected from the histological damage scores.
Assessment of the myelo peroxidase activity in rat colons at day five post DNBS exposure reveals an increase in activity in all three groups with the greatest myelo peroxidase activity observed in the safflower oil group, followed by the canola oil group and the fish oil group. While attempting this procedure, it's important to remember to lightly anesthetize mice and to optimize DNBS doses After its development. This technique paved the way for researchers in the field of inflammatory bowel disease to explore mechanisms that control intestinal inflammation in animal models of IBD.
Don't forget that working with DNBS can be extremely hazardous, so precautions such as protective clothing, gloves, goggles, and facials should always be worn while preparing the solution.
